Acute Comparative Analysis of Anesthetic Methods in Neonatal Mice (Mus musculus) Using Ultrasonic Vocalizations and Biochemical Markers

Hypothermia has been used as an anesthetic technique in neonatal rodents since 1934. While the long-term impacts of neonatal anesthetic exposure in adult mice are still under investigation, its acute effect during the neonatal period is poorly characterized. Although hypothermia can achieve sufficient anesthetic depth to eliminate pain sensation, its effect during induction and recovery remains unclear. Mice emit ultrasonic vocalizations (USVs) within a range of 30-120 kHz for normal behavior and communication, with lower frequencies often associated with pain and distress. Using USV analysis and other physiologic measures, we assessed the well-being of neonatal mice subjected to different anesthetic exposures at different ages (postnatal day [PND] 2, 5, 8, 11, and 14). C57BL/6 neonatal mice (n = 483) were randomly assigned to 1 of 7 groups: no handling or anesthesia, handling only, hypothermia (PND 2, 5, and 8 only), isoflurane vaporizer, sevoflurane vaporizer, isoflurane drop, or sevoflurane drop methods. USVs were recorded at 4 time points: before the anesthetic exposure (T0), immediately during recovery (DR), 5 minutes after recovery (PR), and 120 minutes after recovery (T120) (n ≥ 12 pups per group). Induction and recovery were timed. At T120, terminal blood was collected to measure stress-related biochemical markers (blood glucose, corticosterone, norepinephrine). Our results show that neonatal mice receiving hypothermia anesthesia had significantly lower frequency of USVs during and after recovery, longer duration of USV calls, and longer recovery time compared with other anesthetic groups. Our findings suggest that hypothermia anesthesia induces acute distress in neonatal mice ≤PND 8 compared with inhalational anesthetics. In accordance with the principles of the 3Rs to minimize pain and distress, using inhalant anesthesia (isoflurane or sevoflurane) is preferable to hypothermia for neonatal anesthesia in mouse pups at ≤PND 8.